4.6 | Introduction of fluorescence-labeled PCNDs into cells
To microscopically observe the intracellular accumulation of PCNDs, PCNDs were modified with fluorescence-labeled 9E5 and fluorescence-labeled PEG-lipid. 9E5 was prepared using 9E5-expressing hybridoma cells[31,32] and biotinylated by reacting with biotin-NHS (13 eq), which was synthesized as previously reported[39]. After purification with a gel filtration column, biotinylated 9E5 was fluorescently labeled by allowing it to react with Alexa647-NHS (10 eq). Biotinylated and fluorescently labeled 9E5 was purified by gel filtration and then was conjugated with PCNDs, as previously reported[30,31]. Here, PCNDs were prepared using DPPC, DPPE-PEG-OMe, DPPE-PEG-biotin, and DPPE-PEG-TAMRA. Biotinylated and fluorescently labeled 9E5 was mixed with NeutrAvidin for 15 min at room temperature to prepare the 9E5-NeutrAvidin conjugate. PCNDs were suspended in 9E5-NeutrAvidin solution and incubated for 30 min on ice for modification with 9E5. After centrifugation at 3000 × g for 5 min, the PCND pellet was washed with cold PBS and suspended in RPMI-based culture medium. DLD1 cells were treated with 9E5-conjugated PCND suspension for 3 h under culture conditions. After washing the dish surface, the cells were observed using a confocal laser-scanning microscope (LSM 510 META; Carl Zeiss, Germany) equipped with a 20× objective lens. The fluorescence of TAMRA (Em: 560/610 nm) and AF647 (Em:650/680 nm) was observed using excitation lasers at wavelengths of 552 and 633 nm, respectively.